Enzymes are proteins with biocatalytic activity which exhibit high specificity, i.e., their power to catalyze the reaction of only certain molecules, with large rate accelerations. Although they are large and complex molecules, their power to catalyze reactions can be attributed to only two phenomena, binding and catalysis. Binding not only contributes largely to the specificity of the reaction but by stereochemistry also brings the substrate into close proximity and in the correct orientation to the catalytic site, which is ultimately responsible for the large rate accelerations which operate intramolecularly. Indeed, there are other factors: the microscopic environment of the reaction site; the stabilization of the transition state by hydrogen bonding, etc., which contribute toward the enzymatic activity in different enzymes by varying degrees; but binding, particularly of the transition state, and catalysis are the two essential features of all enzymes.
Enzyme modeling is the science of scientifically mimicking the exact nature of the binding site in terms of shape, size and microscopic environment; as well as the catalytic site in terms of identity of groups, stereochemistry, interatomic distances of various groups and the mechanism of action of the enzyme. Such information about the enzymes can be obtained by amino acid sequencing, CPK modeling, X-ray crystallographic studies, inhibition studies and specificity studies of the enzyme. It is known, for example, that the binding site for chymotrypsin is hydrophobic in nature, 10-12 .ANG. deep and 3.5 to 5.5 to 6.5 .ANG. in cross section, which gives a snug fit to an aromatic ring, also hydrophobic in nature, which is 6 .ANG. wide and 3.5 .ANG. thick. The active site of chymotrypsin has been shown to contain only three amino acids, namely serine 195, histidine 57 and aspartate 102. The unique feature about these amino acids, however, is the functional groups which they carry. They are shown in Table I and consist of a hydroxyl group of serine 195, an imidazole group of histidine 57 and a carboxylate ion of aspartate 102.
TABLE I ______________________________________ The Chymotrypsin Active Site AMINO ACID FUNCTIONALITY ______________________________________ 1. serine 195 hydroxyl 2. histidine 57 imidazolyl 3. aspartate 102 carboxylate ion ______________________________________
The well-known "proton transfer relay" system proposed for the mechanism of action of chymotrypsin consists of two proton transfers, one initiated by the carboxylate ion and the other initiated by the imidazole to increase the nucleophilicity of the hydroxyl oxygen atom of serine toward the carbonyl function of the amide or ester substrate bound in the hydrophobic pocket of the enzyme to give an acyl-enzyme intermediate. Deacylation occurs via the same two proton transfers increasing the nucleophilicity of the hydroxyl group of water, which attacks the carbonyl group (C.dbd.O) of the acyl-enzyme ester.
A model of chymotrypsin should essentially contain a hydrophobic pocket to act as a binding site attached to a hydroxyl group (OH), an imidazole group ##STR2## and a carboxylate ion (CO.sub.2.sup.-) placed at the right distances and correct stereochemistry to participate in a "proton transfer relay" system.
Cramer has disclosed methods of modifying cyclodextrin by imidazole to produce a chymotrypsin model (Angew, Chem. 78, 641 (1966)), and Breslow et al. have disclosed methods of synthesizing models of ribonuclease, transaminase, and a thiazolium dependent enzyme based on cyclodextrin (Breslow, et al., J. Am. Chem. Soc. 100, 3227-3229 (1978); Breslow, et al., J. Am. Chem. Soc. 105, 1390-1391 (1983); and Breslow, et al., Bioorg. Chem., 12, 206-220 (1984)). In both Cramer's and Breslow's models, the catalytic groups are linked to C-6 of cyclodextrin, placing it on the primary edge of the molecule. It is placed in that location in distinction to the secondary (C-2, C-3) faces of the molecules. Apparently, the secondary side is more open and is the preferential locus of binding of large molecules for it is also the face on which the chirality of cyclodextrin is more apparent. The Cramer model is relatively unreactive.